Photodynamic therapy, formulation usable for this purpose, and method for the production and use thereof

ABSTRACT

A treatment of cancer, such as, for example, bladder cancer, uses a formulation that contains sodium hypericinate that is bonded to polyvinylpyrrolidone or complexed with polyvinylpyrrolidone.

FIELD OF THE INVENTION

The invention relates to a method for photodynamic therapy, aformulation of hypericin, which can be used in photodynamic therapy, amethod for the production, and the use thereof for the production of aphotosensitizer.

BACKGROUND OF THE INVENTION

The photodynamic therapy (PDT) is a method that is suitable for treatingtumors and premalignant changes in the skin and mucosa of various holloworgans (Juarranz et al., 2008; Agostinis et al., 2011).

The PDT is based on the interaction of three components:photosensitizer, light in the visible range, and oxygen.

After systemic or topical application of a photosensitizer, anaccumulation of the photosensitizer is carried out in the malignanttissue. Using light of a suitable wavelength, the photosensitizer can bestimulated. In the stimulated state, energy is transferred to areactant, e.g., molecular oxygen. In this case, reactive oxygenmolecules are generated, which in turn damage cellular structures of thetumor tissue, by which cellular processes such as apoptosis and necrosisare introduced (Agostinis, et al., 2011; Allison and Sibata, 2010).

A more ideal photosensitizer for the PDT shows selective accumulation intumor cells, no or minimal systemic toxicity, and it is photochemicallyefficient.

Hypericin 1,3,4,6,8,13-hexahydroxy-10,11-dimethylphenanthro(1,10,9,8-opqra)perylene-7,14-dione was already described as a potentialphotosensitizer in the literature (Agostinis et al., 2002).

In in-vitro studies, the effectiveness of hypericin in PDT was shown ina series of cell lines (Karioti and Bilia, 2010).

Moreover, in-vivo animal studies confirm the potential of hypericin forapplication in PDT (Bhuvaneswari et al., 2010; Chen et al., 2003; Liu etal., 2000; Sanovic et al., 2011).

Hypericin is hydrophobic and water-insoluble. For this reason, in thepast, hypericin was brought into solution using the organic solventdimethyl sulfoxide (DMSO) or a water-soluble polymer, polyethyleneglycol (PEG).

Animal experiments in a rat model showed encouraging results withrespect to the PDT of bladder carcinoma. In this case, hypericin wasbrought into the tumor cells using polyethylene glycol. With a hypericindose of 30 μM and an irradiation with light (595 nm) of an intensity of25 up to 50 mW/cm², up to 98% of the tumor cells were killed (Kamuhabwaet al. 2003).

For a clinical application, however, a water-soluble formulation ofhypericin is required, which has tumor selectivity and can be stimulatedwith light in the visible range.

The document WO 01/89576 A2 describes how the solubility of hypericincan be increased by the adjuvant polyvinylpyrrolidone (povidone, PVP).

The use of PVP-hypericin in PDT is also described in WO 2014/079972 A1.WO 2014/079972 A1 deals with in particular a device that can be used inthe PDT of hollow organs, such as the human bladder.

PVP-hypericin shows a selective accumulation in tumor cells in vitro andin vivo (Kubin et al., 2008; Vandepitte et al., 2011).

SUMMARY OF THE INVENTION

As a first object, the invention is based on making available animproved method for photodynamic therapy.

This object is achieved with a method for photodynamic therapy oftumors, in which as a photosensitizer, a complex or a compound thatconsists of hypericin and a polymeric complexing agent is used and inwhich as a photosensitizer, a complex or a compound that consists of analkali salt of hypericin and a polymeric complexing agent is used.

The object of the invention is also to make available a sterile andstable formulation of hypericin, which can be used for clinicalapplication in photodynamic therapy (PDT).

This object is achieved with a formulation that contains hypericin thatis bonded or complexed to a polymeric complexing agent, wherebyhypericin is present as a salt.

The object of the invention is also to make available a method for theproduction of a formulation of hypericin that can be used in aphotodynamic therapy as a photosensitizer.

This object is achieved with a method in which hypericin salt is bondedor complexed to a polyethylene glycol or to a poly-N-vinyl amide,preferably polyvinylpyrrolidone (PVP).

Moreover, the invention is based on an advantageous use of theformulation according to the invention.

In this respect, the invention relates to the use of the formulationaccording to the invention for the production of a photosensitizer thatcan be used in photophysical or photodynamic therapy.

Preferred and advantageous embodiments of the therapy according to theinvention, the formulation according to the invention, the method forthe production, and the use thereof are subjects of the subclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 depict the survival of tumor cells after treatment withthe hypericin formulation of the invention at a light intensity of 5 and25 mW/cm².

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly enough, it has been shown that the formulation of hypericinaccording to the invention can be applied in a stable manner and thusunder clinical conditions in the therapy according to the invention onlywhen hypericin is present as a salt.

An evaluation of the therapy according to the invention with use of theformulation of hypericin according to the invention in animalexperiments has shown, surprisingly enough, that in the case of a doseof 30 μM of hypericin in a stable formulation with PVP according toExample 1, a required light intensity of 5 or 25 mW/cm² at a wavelengthof 595 nm and a 120-minute exposure time in the bladder (instillationtime) is sufficient to kill 98% of the tumor cells. The same result of98% killed tumor cells is also achieved at the same light intensity and40 μM of hypericin at a 15- or 30-minute exposure time and treatmentwith light of a wavelength of 610 nm. Also, an instillation time of 1hour, with 20 μM of hypericin, equal light intensity, and 570 nm oflight frequency, achieves a kill rate of 97%, and an instillation timeof 120 minutes, with 9 μM of hypericin, equal light intensity, andtreatment at 600 nm, achieves a kill rate of 95%. Thus, in the case oflight intensities of 5 to 25 mW/cm² with light frequencies of 570 to 610nm, hypericin concentrations of 9 to 40 μM, and exposure times ofbetween 15 and 120 minutes, a kill rate of 95 to 98% of the tumor cells(Practical Examples 1, 2, 3, and 4) is achieved.

The effectiveness of a PDT is essentially dependent upon the totalamount of light. At the same time, the probability of local side effectsis increased with increasing light intensity.

Using therapy according to the invention, in particular with use of theformulation according to the invention, an improved accumulation inmalignant tissue is achieved, by which a considerably reduced lightintensity of already 5 to at most 25 mW/cm² is sufficient in order tokill tumor cells.

The selective concentration of the formulation of hypericin according tothe invention and the surprisingly low light intensity, which wasrequired for a PDT in the animal model during application of theformulation of hypericin according to the invention, allows theapplication in the treatment of lesions in various cavities of the bodythat can be reached with the necessary light dose.

Below, examples of the formulation of hypericin (hypericin-PVP complex)according to the invention are presented.

General Procedure for the Production of a Formulation with the ActiveIngredient Sodium Hypericinate:

The goal is the production of a hypericin-containing formulation forapplication as a photosensitizer in the field of photodynamic therapy.

The formulation according to the invention is produced from a hypericinsalt, in particular from sodium hypericinate.

In order to define the hypericin content of the starting material, inaddition to the determination of contents, primarily water content and,in the case of sodium hypericinate, the proportion of sodium arespecified.

The chemical-physical properties can have an influence on theformulation of the pharmaceutical agent.

For the clinical application, a stability of the formulation accordingto the invention is necessary. The stability is ensured through thecomposition of the finished product and at the same time also relates tothe production method. Because of the buffer systems used, adequatestability of the bulk solution can also be achieved during productionuntil lyophilization of the finished product takes place.

As buffer systems, various additives can be used, which preferably bothfor the bulk solution and for the reconstituted solution achieve aphysiologically compatible pH and an osmotic pressure of 290 mOsmol/kgafter reconstitution with 50 ml of water for injection. Phosphate orcitrate buffer systems can be used primarily.

After the bulk solution is made up from the above-mentioned components,the corresponding amount of the bulk solution is decanted into injectionflasks and freeze-dried.

Example 1

From sodium hypericinate, a solution with a target weighed-in amount of90.0 mg of hypericin is produced.

5.0 g of the hypericin solution is added to 1,875 mg of PVP k25 andcompletely dissolved.

This solution is quantitatively made up to 250.0 g with a phosphatebuffer solution. The final concentration of this solution is 0.0225 mgof hypericin/g of solution.

For lyophilization, a defined amount of the thus obtained bulk solutionis decanted into injection flasks, and the finished lyophilizate isproduced with a corresponding lyo program.

Example 2

The procedure is the same as indicated in Example 1, whereby instead ofPVP k25, PVP k17 is used for complexing sodium hypericinate.

Example 3

The procedure is the same as indicated in Example 1, whereby instead ofPVP k25, PVP k30 is used for complexing sodium hypericinate.

Example 4

The procedure is the same as indicated in Examples 1, 2, or 3, wherebyinstead of the phosphate buffer solution, a citric acid buffer solutionis used.

The bulk solutions that are produced as described in Examples 1 to 4 canbe produced with different hypericin contents.

The effectiveness of the therapy according to the invention with use ofthe formulation of hypericin according to the invention was examined ina preclinical study with use of the formulation as Example 1.

Practical Examples

To this end, the formulation of hypericin according to the invention forthe PDT was studied in a preclinical, orthotopic bladder tumor model inrats. In all examples, the tumors were treated with the formulation ofhypericin according to the invention in different concentrations of 9 to40 μM, with different light intensities of 5 or 25 mW/cm², differentlight frequencies of 570 to 610 nm, and different instillation times.

Example 1

After a 2-hour instillation with 30 μM of the formulation of hypericinaccording to the invention and different light intensities (5 or 25mW/cm²) with light of a wavelength of 595 nm, up to 98% of the tumorcells were killed.

Example 2

After a 1-hour instillation with 20 μM of the formulation of hypericinaccording to the invention and different light intensities (5 or 25mW/cm²) with light of a wavelength of 570 nm, up to 97% of the tumorcells were killed.

Example 3

After a 15- or 30-minute instillation with 40 μM of the formulation ofhypericin according to the invention and different light intensities (5or 25 mW/cm²) with light of a wavelength of 610 nm, up to 98% of thetumor cells were killed.

Example 4

After a 2-hour instillation with 9 μM of the formulation of hypericinaccording to the invention and different light intensities (5-25 mW/cm²)with light of a wavelength of 600 nm, up to 95% of the tumor cells werekilled.

The results of the studies on the rat model are presented in the figure.In the diagrams, “ns” stands for “not significant,” and “*” stands for“significant.” The diagrams of the figure show the survival of tumorcells after treatment with the formulation of hypericin according to theinvention and light. 24 hours after the treatment, the bladder tissuewas dissociated, and the surviving cells were determined using aclonogenic assay in comparison to the control (without PVP-hypericin andlight).

The relative survival of the cells under PDT conditions (PVP-hypericinaccording to Example 1 and treatment with light) is (depicted as meanvalue+SD): 7.4 (+/−6.4)% with use of 5 mW/cm² and 2.4 (+/−4.0)% at 25mW/cm² and a treatment period with light of 60 minutes. This is depictedin two diagrams in the figure.

REFERENCES

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1. Method for photodynamic therapy of tumors, whereby as aphotosensitizer, a complex or a compound that consists of hypericin anda polymeric complexing agent is used, characterized in that as aphotosensitizer, a complex or a compound that consists of an alkali saltof hypericin and a polymeric complexing agent is used.
 2. Methodaccording to claim 1, wherein the alkali salt of hypericin is sodiumsalt or potassium salt.
 3. Method according to claim 1, wherein aphotodynamic therapy of cancer, in particular bladder cancer, is made.4. Method according to claim 1, wherein the photosensitizer withconcentrations of 9 to 40 μM of hypericin is used.
 5. Method accordingto claim 1, wherein the photosensitizer is used with light intensitiesof 5 to 25 mW/cm² at wavelengths of the visible light in theyellow/orange/red spectral range.
 6. Method according to claim 1,wherein instillation times of 15 to 120 minutes are used in order tokill more than 90% of the tumors.
 7. Method according to claim 1,wherein the complexing agent is a polyethylene glycol or a poly-N-vinylamide.
 8. Method according to claim 7, wherein poly-N-vinyl amide is apolyvinylpyrrolidone (PVP) of various degrees of polymerization andcross-linking.
 9. Method according to claim 8, wherein thepolyvinylpyrrolidone is PVP k17, PVP k25 or PVP k30.
 10. Formulation forphotodynamic therapy containing hypericin that is bonded or complexed toa polymeric complexing agent, wherein the hypericin is present as asalt.
 11. Formulation according to claim 10, wherein the complexingagent is a polyethylene glycol or a poly-N-vinyl amide.
 12. Formulationaccording to claim 11, wherein poly-N-vinyl amide is apolyvinylpyrrolidone (PVP) of various degrees of polymerization andcross-linking.
 13. Formulation according to claim 12, wherein thepolyvinylpyrrolidone is PVP k17, PVP k25 or PVP k30.
 14. Formulationaccording to claim 10, wherein the hypericin salt is an alkali metalsalt, in particular potassium salt or sodium salt.
 15. Method for theproduction of a formulation according to claim 10, wherein the hypericinsalt is bonded or complexed to a polyethylene glycol or to apoly-N-vinyl amide, preferably polyvinylpyrrolidone (PVP).
 16. Methodaccording to claim 15, wherein the complexing is performed in aqueous,optionally buffered, solution.
 17. Method according to claim 15, whereinthe formulation is prepared for intravenous, intracavity, inhalative,oral, intraperitoneal and topical administration, in hydrophilic orhydrophobic vehicles, preferably in the form of a solution, a cream, agel, an aerosol, emulsions or as a patch.
 18. Method according to claim16, wherein the aqueous solution is decanted into injection flasks andfreeze-dried.
 19. Method according to claim 2, wherein a photodynamictherapy of bladder cancer is made.
 20. Method according to claim 2,wherein the photosensitizer with concentrations of 9 to 40 μM ofhypericin is used with light intensities of 5 to 25 mW/cm² atwavelengths of the visible light in the yellow/orange/red spectralrange.